Effects of enzyme types and hydrolysis time on the production of antioxidant peptides from Spirulina platensis

Document Type : Research Article


1 MS student of Food Science and Technology, Shahr-e-Qods Branch, Islamic Azad University, Tehran,Iran

2 Assistant Professor of Food Science and Technology, Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran

3 Associate professor, Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST),


Conditions of enzymatic hydrolysis including time, enzyme types, and degree of hydrolysis is impressive on biological activities of the protein hydrolysate. The aim of the present research was to consider the effect of pepsin, chymotrypsin and combination of two enzymes (E/S:10/1, 5 hr, 37 °C), also the time and degree of hydrolysis on the production of antioxidant peptides from Spirulina platensis protein that was extracted through sonication treatment ( 200 W, 20 KHz, 5 min). The progress of enzymatic hydrolysis and antioxidant activity were considered‏ through the time of hydrolysis by respectively, O-phethaldialdehyde (OPA) and DPPH and ABTS radicals scavenging assays. The content of free amino groups reached from 3.272 to respectively, 3.653, 4.552, 3.713 µM Leu/mg protein for samples hydrolyzed by pepsin, chymotrypsin, and combined enzymes. Antioxidant activity increased by the progress of hydrolysis and finally DPPH and ABTS radicals scavenging activity increased to 23 and 110 after 150 min hydrolysis by pepsin, and to 18 and 159 after 120 min hydrolysis by chymotrypsin. Hydrolysis by combined enzymes Increased DPPH and ABTS radicals scavenging activities to respectively, 25 µM TE/mg protein ( after 90 min) and 230 µM TE/mg protein (after 210 min). Altogether, Our results confirmed the production of antioxidant peptides through pepsin and chymotrypsin hydrolysis of. Also, our results showed that chymotrypsin was more effective compared to pepsin and a combination of two enzymes was more effective for producing antioxidant peptides from Spirulina platensis.

Graphical Abstract

Effects of enzyme types and hydrolysis time on the production of antioxidant peptides from Spirulina platensis


  • Enzymatic hydrolysis by  gastrointestinal enzymes improved the antioxidant activity of protein extracted from Spirulina microalgae.
  • Chymotrypsin was the most effective enzyme in the progress of enzymatic hydrolysis and producing antioxidant peptides from Spirulina microalgae.
  • A direct relationship was observed between the progress of enzymatic hydrolysis and antioxidant activity of protein hydrolysate, in most cases.


Main Subjects

[1]     Butcher, H. K., Bulechek, G. M., Dochterman, J. M. M., & Wagner, C. (2018). Nursing Interventions classification (NIC)-E-Book. Elsevier Health Sciences.
[2]     Salehifar, M., Shahbazizadeh, S., Khosravi-Darani, K., Behmadi, H., & Ferdowsi, R. (2013). Possibility of using microalgae Spirulina platensis powder in industrial production of Iranian traditional cookies. Iranian Journal of Nutrition Sciences & Food Technology, 7(4), 63-72.
[3]     Volkmann, H., Imianovsky, U., Oliveira, J. L., & Sant'Anna, E. S. (2008). Cultivation of Arthrospira (Spirulina) platensis in desalinator wastewater and salinated synthetic medium: protein content and amino-acid profile. Brazilian Journal of Microbiology, 39(1), 98-101.
[4]     Belay, A., & Gershwin, M. E. (2007). Spirulina (Arthrospira). In Spirulina in Human Nutrition and Health (pp. 11-35). CRC Press.
[5]     Gupta, M., Dwivedi, U. N. & Khandelwal, S. 2011. C-Phycocyanin: An effective protective agent against thymic atrophy by tributyltin. Toxicology Letters, 204: 2–11. 22
[6]     Vijayabaskar, P., & Shiyamala, V. (2012). Antioxidant properties of seaweed polyphenol from Turbinaria ornata (Turner) J. Agardh, 1848. Asian Pacific Journal of Tropical Biomedicine, 2(1), S90-S98.
[7]     Yokozawa, T., Kim, H. Y., Nonaka, G. I., & Kosuna, K. (2002). Buckwheat extract inhibits progression of renal failure. Journal of agricultural and food chemistry, 50(11), 3341-3345.Wang, J., Zhang, Q., Zhang, Z., & Li, Z. (2008). Antioxidant activity of sulfated polysaccharide fractions extracted from Laminaria japonica. International Journal of Biological Macromolecules, 42(2), 127-132.
[8]     Chen, H. M., Muramoto, K., Yamauchi, F., & Nokihara, K. (1996). Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. Journal of agricultural and food chemistry, 44(9), 2619-2623.
[9]     Yogianti, F., Kunisada, M., Nakano, E., Ono, R., Sakumi, K., Oka, S., ... & Nishigori, C. (2014). Inhibitory effects of dietary Spirulina platensis on UVB-induced skin inflammatory responses and carcinogenesis. Journal of Investigative Dermatology, 134(10), 2610-2619.
[10] Mechmeche, M., Kachouri, F., Ksontini, H., & Hamdi, M. (2017). Production of bioactive peptides from tomato seed isolate by Lactobacillus plantarum fermentation and enhancement of antioxidant activity. Food Biotechnology, 31(2), 94-113.
[11] Lisboa, C. R., Pereira, A. M., & Costa, J. A. V. (2016). Biopeptides with antioxidant activity extracted from the biomass of Spirulina sp. LEB 18. African Journal of Microbiology Research, 10(3), 79-86.
[12] Kuddus, M., Singh, P., Thomas, G. and Al- Hazimi, A., 2013. Recent developments in production and biotechnological applications of C- Phycocyanin. BioMed Research International, Article ID 742859, 9P.
[13] Mirzaei, M., Mirdamadi, S., Ehsani, M. R., Aminlari, M., & Hosseini, E. (2015). Purification and identification of antioxidant and ACE-inhibitory peptide from Saccharomyces cerevisiae protein hydrolysate. Journal of Functional Foods, 19, 259-268.
[14] Kang, K. H., Qian, Z. J., Ryu, B., & Kim, S. K. (2011). Characterization of growth and protein contents from microalgae Navicula incerta with the investigation of antioxidant activity of enzymatic hydrolysates. Food Science and Biotechnology, 20(1), 183-191. (Hartree, 1972).
[15] Heo, S. J., Park, E. J., Lee, K. W., & Jeon, Y. J. (2005). Antioxidant activities of enzymatic extracts from brown seaweeds. Bioresource Technology, 96(14), 1613-1623.
[16] Sheih, I. C., Wu, T. K., & Fang, T. J. (2009). Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems. Bioresource Technology, 100(13), 3419-3425.
[17]            شیخ نژاد، ع.، لباب پور، ع.ا. و م. ن. (1394). اﻓﺰاﻳﺶ ﺗﻮﻟﻴﺪ ﺑﺎﻛﺘﺮی ﺳﻴﺎﻧﻮ  اﺳﭙﻴﺮوﻟﻴﻨﺎ  ﺑﺎﻛﻨﺘﺮل ﻫﻢ زدنﺷﻴﻤﻴﺎﻳﻲ و ﺗﺮﻛﻴﺐ  ﻛﺸﺖ ﻣﺤﻴﻂ. مجله پژوهش های گیاهان (مجله زیست شناسی ایران). جلد 28. شماره 2. صفحات: 353-344.
[18] JAO, C. L., & KO, W. C. (2002). 1, 1‐Diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging by protein hydrolyzates from tuna cooking juice. Fisheries Science, 68(2), 430-435.
[19] He, R., Girgih, A. T., Malomo, S. A., Ju, X., & Aluko, R. E. (2013). Antioxidant activities of enzymatic rapeseed protein hydrolysates and the membrane ultrafiltration fractions. Journal of Functional Foods, 5(1), 219-227
[20] Shahidi, F., & Zhong, Y. (2008). Bioactive peptides. Journal of AOAC international, 91(4), 914-931.
[21] Baratzadeh, M. H., Asoodeh, A., & Chamani, J. (2013). Antioxidant peptides obtained from goose egg white proteins by enzymatic hydrolysis. International Journal of Food Science & Technology, 48(8), 1603-1609.
[22] Wang, Q., Huang, Y., Qin, C., Liang, M., Mao, X., Li, S., . . . Ma, C. W. (2016). Bioactive Peptides from Angelica sinensis Protein Hydrolyzate Delay Senescence in Caenorhabditis elegans through Antioxidant Activities. Oxidative medicine and cellular longevity, 2016.
[23] Samaranayaka, A. G., & Li-Chan, E. C. (2008). Autolysis-assisted production of fish protein hydrolysates with antioxidant properties from Pacific hake (Merluccius productus). Food Chemistry, 107(2), 768-776.
[24]  Byun, H. G., Lee, J. K., Park, H. G., Jeon, J. K., & Kim, S. K. (2009). Antioxidant peptides isolated from the marine rotifer, Brachionus rotundiformis. Process Biochemistry, 44(8), 842-846.
[25] Bhaskar, N., Benila, T., Radha, C., & Lalitha, R. G. (2008). Optimization of enzymatic hydrolysis of visceral waste proteins of Catla (Catla catla) for preparing protein hydrolysate using a commercial protease. Bioresource Technology, 99(2), 335-343.
[26] JAO, C. L., & KO, W. C. (2002). 1, 1‐Diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging by protein hydrolyzates from tuna cooking juice. Fisheries Science, 68(2), 430-435.
[27] Fruton, J. S. (2014). Specificity and mechanism of pepsin action. Paper presented at the Structure–Function Relationships of Proteolytic Enzymes: Proceedings of the International Symposium, Copenhagen June 16-18, 1969, No. 37 in the Series of the International Union of Biochemistry Sponsored Symposia.
[28] Blow, D. M. (1976). Structure and mechanism of chymotrypsin. Accounts of chemical research, 9(4), 145-152.
[29] Kim, S. K. (Ed.). (2013). Marine proteins and peptides: biological activities and applications. John Wiley & Sons.